Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD50 sensitivity to 30 µM 4-HNE and 100 µM H2O2 at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H2O2 resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease

Oxidative adducts are enhanced in experimental and human myocardium during Chagas disease.

<p>(<b>A</b>) Sprague-Dawley rats (or C3H/HeN mice) were infected with <i>T. cruzi,</i> and cells were harvested at day 40 (acute stage) and 180 (chronic stage) post-infection. Heart homogenates were resolved on 10% acrylamide gels, and Western blotting was performed with specific antibodies to detect 4 hydroxynonenal (4-HNE, <i>panel a</i>), malondialdehyde (MDA, <i>panel b</i>), dinitrophenyl (DNP)-derivatized carbonyl (<i>panel c</i>), and 3-nitrotyrosine (3NT, <i>panel d</i>) adducts. Coomassie blue staining of membranes (<i>panel e</i>) confirmed the equal loading of samples. (<b>B</b>) Cryostat sections of human cardiac biopsies (5-µm) from normal healthy donors (<i>panels a, c, e</i>) and chagasic patients (<i>panels b, d, f</i>) were submitted to immunohistochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028449#s2" target="_blank">Materials and Methods</a>. Shown are representative images of immunostaining with anti-4-HNE antibodies (<i>panels a, b</i>). Tissue sections were incubated with DNPH to derivatize carbonyl proteins, and immunostaining was performed with anti-DNP antibody (<i>panels c–f</i>). HRP-conjugated (<i>panels a–d</i>) and rhodamine-conjugated (<i>panels e, f</i>) secondary antibodies were utilized to capture the color (brown) or fluorescence signal, respectively.</p

Oxidized titin exhibited increased immunogenicity in chagasic patients.

<p>Recombinant titin was <i>in vitro</i> oxidized with 4-HNE (30 µM) or H₂O₂ (100 µM). An enzyme-linked immune-sorbent assay (ELISA) was performed to titer the antibody level against recombinant and oxidized titin in sera samples of normal healthy controls, seropositive chagasic patients, and seronegative subjects with cardiomyopathy of other etiologies (OCM). n = 25/group (*<i>p</i><0.05).</p

Oxidative stress affects cardiomyocyte viability.

<p>Human cardiomyocytes were incubated with (<b>A</b>) 4-hydroxynonenal (0–40 µM) or (<b>B</b>) H₂O₂ (0–200 µM) for 0, 2, 6, 12, and 24 h. An MTT assay was performed to examine cell viability (*<i>p</i><0.05).</p

Identification of proteins recognized by immune sera from chagasic patients.

<p>Human cardiomyocyte lysates were treated with 100-µM H₂O₂ or 30-µM 4-hydroxynonenal (4-HNE). Cell lysates were resolved by 2 dimensional gel electrophoresis and probed with sera samples from normal subjects, seropositive chagasic patients that exhibited no clinical disease (CD0–1) or mild-moderate symptoms of clinical disease (CD2–3), and seronegative other cardiomyopathy (OCM) patients. Protein spots recognized by immune sera were submitted to MALDI-TOF-MS/MS analysis. A −, +, ++, and +++ indicates none, mild, moderate and strong recognition of protein spots by immune sera. Gels were also probed with anti-dinitrophenyl (DNP) and anti-4-HNE antibodies. In column 1, * and ^# indicate protein spots that were recognized by anti-HNE and anti-DNP antibodies, respectively.</p